The invention relates to protocols for the treatment of tumors. More specifically, the invention concerns insertion of a methioninase gene into tumor cells, optionally in combination with administration of methioninase, methionine deprivation or administration of a chemotherapeutic agent.
Various approaches to tumor treatment using gene replacement or other genetic therapy protocols have been suggested and verified in animal models. For example, since it is known that the p53 gene is a tumor suppressor (Harris, C. C., JNCI (1996) 88:1442-1445), the antitumor effects of p53 gene transfer have been explored. Successful results for tumors which contain defective p53 genes have been shown by Roth, J. A., et al., Nat Med (1996) 2:985-991 using retrovirus-mediated gene transfer in patients with lung cancer. Adenoviral vectors encoding p53 for gene therapy have been described by Wills, K. N., et al., Hum Gene Ther (1994) 5:1079-1088. An animal model of malignant human breast cancer in nude mice was shown to respond to adenoviral-mediated delivery of p53 by Lesoon-Wood, L. A., et al., Hum Gene Ther (1995) 6:395-405.
Alternatively, the Herpes simplex virus thymidine kinase gene (HSV-tk) in combination with gancyclovir has been shown to inhibit tumor growth in melanoma mode by Vile, R., et al. Cancer Res Dec. 1 (1994); 54(23):6228-34. However, surrounding cells may also be killed using such protocols in a xe2x80x9cbystanderxe2x80x9d effect.
It is now well known that most tumor cells, in contrast to normal cells, are dependent on an external supply of methionine due to a high demand for methylation reactions. Thus, more methionine is required than is available from the endogenous synthesis route from homocysteine. Cancer cells arrest when exogenous methionine is unavailable in the late S/G2 phase of the cell cycle (Hoffman, R. M., et al., Proc Natl Acad Sci USA (1980) 77:7306-7310.
Deprivation of methionine in the diet has been shown both in animal models and in the clinic to extend survival time (Breillout, F., et al., JNCI (1990) 82:1628-1632; Goseki, N., et al, Cancer (1992) 69:1865-1872; Hosiya, Y., et al., Anticancer Res (1996) 16:2719-2724; Guo, H., et al., Cancer Res (1993) 53:5676-5679; Goseki, N., et al., Japan J Cancer Res (1995) 86:484-489). In addition, administration of recombinant methioninase (rMETase) has been found to have antitumor efficacy both in vitro and in vivo (U.S. Pat. No. 5,690,929; Tan, Y., et al., Anticancer Res (1996) 16:3931-3936; Yoshioka, T., et al., Cancer Res (1998) 58:5283-5287). Methioninase is also known to deplete homocysteine levels, thus reducing the concentration of the endogenous precursor for methionine (U.S. Pat. No. 5,715,835).
It has now been found that the efficacy of methionine reduction protocols, including treatment with methioninase, can be enhanced and improved by introducing an expression system for the methioninase gene into tumor cells. Thus, the present invention is directed to gene therapy for tumor cell inhibition by supplying the METase gene.
The invention provides a distinct approach to antitumor therapy. Tumor cells are provided an expression system for methioninase, thus permitting an endogenous mechanism for further depleting needed methionine. The expression system can be utilized as the sole method of treatment, but is preferably used in combination with administration of METase protein, methionine depletion in the diet, and/or administration of a chemotherapeutic agent, such as 5-fluorouracil.
Accordingly, in one aspect, the invention is directed to a method to inhibit tumor growth which method comprises treating the cells of said tumor with a composition comprising a nucleic acid molecule, which nucleic acid molecule comprises; in a preferred embodiment, an expression system for a protein having methioninase activity along with a means for effecting the entry of said nucleic acid molecule into the tumor cells. The tumor cells may reside in a vertebrate animal; under those circumstances, the composition is a pharmaceutical or veterinary composition applied to the vertebrate. Preferred vertebrate subjects are mammals, such as primates, including humans, domestic animals, and rodents as well as avians, such as chickens, ducks and geese. The administration of the pharmaceutical or veterinary composition may be supplemented with administration of a protein with methioninase activity and/or placing the animal under a methionine-depleted diet and/or administering to the animal a chemotherapeutic agent.
In another aspect, the invention is directed to tumor cells modified to contain an expression system for production of a protein with methioninase activity. In another aspect, the invention is directed to a nonhuman animal comprising the modified tumor cells. The invention is also directed to adenoviral vectors which comprise the nucleotide sequence encoding methioninase.
In still other aspects, the invention is directed to a method to monitor the expression of methioninase in tumor cells which method comprises assessing the fluorescence of tumor cells that have been treated with an expression system for a fusion protein, said fusion protein comprising an amino acid sequence conferring methioninase activity linked to an amino acid sequence which effects emission of fluorescent light. The fluorescence may be observed in vitro or in vivo, even in the live animal.
The invention provides uniquely effective protocols and materials for treatment of methionine-dependent tumors, as well as assay systems for monitoring the production of METase in tumor cells or in cells generally. The treatment method is selective for tumor cells, since tumor cells are methionine-dependent, whereas normal cells are not.
The expression systems employed in the present invention generally comprise a nucleotide sequence encoding a protein with methioninase activity operably linked to sequences which effect expression of the coding sequence. Alternatively, vectors for delivery of the nucleotide sequence encoding methioninase may be supplied which effect the insertion of the nucleotide sequence into the genome of the host, thus employing the endogenous control sequences to effect expression. The nucleotide sequence is, in this case, coupled to sequences which effect homologous recombination at known targets in the genome, or, alternatively, random integration into the genome may be relied upon. The inclusion of the expression system in a retroviral vector will serve this purpose as well. Use of viral vectors containing an expression system for the methioninase protein is a preferred means of administration.
Since tumor cells are rapidly dividing cells, retroviral vectors can conveniently be used to deliver the methioninase gene. However, other methods for transfecting such cells may also be employed. For example, Herpes simplex vectors can be employed for this purpose. Wolfe, J. H., et al., Nature Genetics (1992)1:379-384. A particularly preferred viral vector is based on adenovirus. Particularly preferred are adenovirus of Type 2 or Type 5 (Ad2 or Ad5). These viruses are not oncogenic and are relatively stable and easy to manipulate. The complete genomes of these types of adenovirus are known and there are a number of known mutants that have been made available for genetic therapy in general. Berkner, K. L., Biotechniques (1988) 6:612-629. However, while viral-based vectors are preferred, any suitable vector for transformation can be employed.
If viral vectors are used, the specificity of infectivity can be enhanced by including receptor-binding moieties in the vector. It is known in the art to modify viral surface molecules with binding domains of ligands, such as erythropoietin which targets erythroleukemia, heregulin which targets breast cancer and neurotensin which targets the colon.
If the vector contains an expression system suitable promoters and enhancers can be used. General constuitive promoters such as SV40 or CMV promoters can be included, along with their enhancer elements, or tissue-specific promoters may be used to enhance specificity. For example, the a fetal protein (AFP) promoter is specific to liver cells and is thus useful when the target tumor is a hepatoma; prostate-specific antigen (PSA) promoter is specific for prostate gland and thus suitable for administration to treat prostate cancer; the carcinoembryonic antigen (CEA) promoter is more general and is effective in cancers of the pancreas, lung, breast and colon.
Means to construct suitable vectors for delivery of the methioninase gene along with provision for its expression are well known in the art.
In order to effect the modification of tumor cells for production of methioninase, the expression system or integrating encoding nucleotide sequence must be formulated so as to enter the cell. Integration of the desired nucleotide sequences into viral vectors, such as adenovirus provides this means of entry. However, retroviral vectors, or other mediators of cellular uptake, such as lipids, or various liposomal type compositions or emulsions may also be employed.
The composition, comprising a nucleotide sequence encoding a protein with methioninase activity, preferably operably linked control sequences, or sequences effecting genomic integration, thus also contains a means for effecting cellular uptake. This composition is supplied to tumor cells, either in vitro or in vivo. In vitro administration is particularly helpful in assessing, in advance, the efficacy of the therapeutic approach for a particular individual, as well as expression levels of the methioninase in the tumor cells. For the latter purpose, it is advantageous to employ the protein having methioninase activity as a fusion protein to a reporter amino acid sequence, most preferably an amino acid sequence which confers fluorescence on the fusion protein. The use of green fluorescent protein (GFP) to confer fluorescence on a fusion protein is well understood in the art; see, for example, Chalfie, M., et al., Science (1994) 263:802-805. The use of green fluorescent protein to monitor metastasis of tumor cells in living subjects is described in U.S. Ser. No. 08/848,539 filed Apr. 28, 1997, now U.S. Pat. No. 6,232,523, and incorporated herein by reference.
While effecting endogenous expression of METase in tumor cells may be used alone if sufficient levels of expression are obtained, the protocol is most effective when combined with additional therapeutic approaches, such as administration of methioninase per se, administration of a methionine-depleted diet, and/or an additional chemotherapeutic agent. Suitable chemotherapeutic agents include, for example, 5-fluorouracil, leucouorin, cisplatin, vinca alkaloids, taxanes and nitrosoureas. A variety of chemotherapeutic agents is well known in the art.
Techniques for administering methioninase itself, for providing a methionine-depleted diet, and for providing chemotherapeutic agents are, of course, well known in the art.
The following examples are intended to illustrate but not to limit the invention.
Retroviral vectors comprising expression systems for either METase-GFP fusion protein or, as a control, GFP per se, were constructed. pLXSN (Clontech, Palo Alto, Calif.) was used as the host retroviral expression vector; the sequence encoding GFP was obtained from pEGFPC-3 described by Chalfie, M., et al., Science (1994) 263:802-805. The METase gene from Pseudomonas putida was obtained from pAC1 described in Tan, Y., et al., Protein Expr. Purif (1997) 9:233-245. The construction was as follows:
The nucleotide sequence encoding METase was amplified by PCR from pAC1 using the primers
5xe2x80x2-CCGCTCGAGATGCACGGCTCCAACAAGCTCCCA-3xe2x80x2 (SEQ ID NO:1)
5xe2x80x2-CGCGGATCCATTAGGCACTCGCCTTGAGTGCCTG-3xe2x80x2 (SEQ ID NO:2)
The resulting 1.2 kb METase encoding DNA was ligated into pEGFPC-3 downstream of the XhoI/BamHI site. The resulting plasmid was digested with NheI, blunt ended, digested by BamHI and ligated into pLXSN which had been digested with HpaI, blunt ended and digested with BamHI. In the resultant vector, pGFP-METase, the GFP-METase gene was driven by the Moloney murine leukemia virus long terminal repeat (LTR) and a neomycin-resistance gene (NeoR) was driven by the simian virus 40 (SV40) early promoter.
The control vector, pGFP, was constructed by inserting the EGFP gene obtained from pEGFP-1 (Clontech) into the pLXSN retroviral vector. Briefly, pEGFP-1 was digested with NotI, blunted by Klenow polymerase and digested with EcoRI; the EGFP gene was separated by purified by low melting agarose electrophoresis. The pLXSN retroviral vector was digested with XhoI, blunted with Klenow and digested with EcoRI. The EGFP gene was then ligated into the digested vector to obtain pGFP.
NIH-3T3 based packaging cells expressing the 10A1 viral envelope, designated PT67 (Clontech, Palo Alto, Calif.), were cultured in DMEM/10% FBS/Pen/Strep. The resulting cells, grown to 70% confluence in 6-well culture dishes, were transfected with DOTATTM (Boehringer-Manheim) liposomal reagent containing saturating amounts of either pGFP or pGFP-METase. The medium was replaced after 18 hours and stable virus-producing cells were selected by culturing for two weeks in the presence of 0.8 mg/ml G418 beginning two days after transfection. The supernatants contain the desired viral vectors designated vGFP-MET or vGFP, respectively.